Alteration of alpha-spectrin ubiquitination after hemorrhagic shock.
BACKGROUND
RBC deformability after trauma and hemorrhagic shock (T/HS) leads to the microcirculatory dysfunction and clinical manifestations of organ failure. However, the cellular mechanism of this phenomenon remains unknown. Spectrins are important for the shape and physical properties of erythrocytes, such as deformability and resistance to mechanical stress. Previous studies have shown that erythrocyte alpha-spectrin is ubiquitinated. Studies of sickled cells and aged erythrocytes, 2 conditions known to have decreased RBC deformability, have shown decreased alpha-spectrin ubiquitination, which may contribute to the inability of these cells to change shape. It was hypothesized that decreased alpha-spectrin ubiquitination could participate in the mechanism(s) whereby T/HS erythrocytes become less deformable.


METHODS
The level of alpha-spectrin ubiquitination in erythrocytes isolated from T/HS rats was determined and compared with erythrocytes from control sham-shocked (T/SS) animals. After T/SS (n = 4) or T/HS (n = 7), alpha- and beta-spectrin subunits were isolated using a low ionic-strength buffer at 37 degrees C for 30 minutes. The relative amount of ubiquitinated alpha-spectrin was evaluated by Western blotting using a monoclonal antibody to ubiquitin.


RESULTS
The relative level of alpha-spectrin ubiquitination (normalized to total alpha-spectrin in the same preparation) was found to be significantly decreased after T/HS (.319 +/- .03) compared with T/SS control erythrocytes (.485 +/- .06, P < .05). To evaluate the content and relative amounts of the other membrane proteins, the profiles of T/HS and T/SS erythrocytes were compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. This did not reveal any significant quantitative differences between T/SS and T/HS spectrin or other membrane proteins.


CONCLUSIONS
The finding of decreased alpha-spectrin ubiquitination after T/HS suggests that this mechanism could contribute to increased rigidity of the erythrocyte membrane.
Caprio
Kimberly
K
Veterans Administration New Jersey Health Care System, East Orange, NJ, USA.
Condon
Michael R
MR
Deitch
Edward A
EA
Xu
Da-Zhang
DZ
Feketova
Eleonora
E
Machiedo
George W
GW
D000704:Analysis of Variance; D000818:Animals; D015153:Blotting, Western; D004195:Disease Models, Animal; D004907:Erythrocyte Deformability; D004912:Erythrocytes / Q000378:metabolism* / Q000648:ultrastructure; D005434:Flow Cytometry / Q000379:methods; D008297:Male; D008855:Microscopy, Electron, Scanning; D010084:Oxidation-Reduction; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012771:Shock, Hemorrhagic / Q000378:metabolism* / Q000503:physiopathology; D013049:Spectrin / Q000378:metabolism*; D054875:Ubiquitination
D013049:Spectrin